Nucleic acid elution

ABSTRACT

This invention relates to the storage on a solid matrix of genetic material, in particular DNA that has been purified prior to the application to the solid matrix. More specifically, the invention relates to a solid matrix for the storage of purified DNA, which matrix has been treated with a solution comprising plant polysaccharide inulin. One advantage of the invention is that an increased amount of DNA can be stored in the solid matrix of the present invention.”

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/519,391, filed Jun. 27, 2012, abandoned, which claims thepriority benefit of PCT/EP2010/070389, filed Dec. 21, 2010, which claimsthe priority benefit of U.S. Provisional Application No. 61/290,652,filed Dec. 29, 2009, which applications are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to the storage on a solid matrix of geneticmaterial, in particular DNA that has been purified prior to theapplication to the solid matrix.

BACKGROUND OF THE INVENTION

Storing of nucleic acids, particularly DNA, is typically accomplished insolution using refrigeration either at 4° C. for up to several days or−20° C. or even lower temperatures for longer periods. This is costlyand space consuming for static storage. It presents greater issues whennucleic acid samples require transportation and samples are oftenshipped in dry ice.

Burgoyne (WO 90/03959) described a method whereby biological samples,usually blood, could be applied to a solid matrix which combinedreagents which lysed the cells. The released DNA was retained on thesolid matrix. These samples could be stored for long periods at roomtemperature.

U.S. Pat. No. 5,939,259 and WO 03/016552 describe techniques whereby theDNA associated with the solid matrix could be eluted for further study.In many cases however, recovery of the purified DNA applied to the solidmatrix results in about 40% of the DNA recovered as determined byquantitative real time PCR. There is clearly a need for a simple methodwhich stores DNA at room temperature for long periods of time but allowsgreater recovery of the applied DNA.

SUMMARY OF THE INVENTION

It has been surprisingly observed that the addition of inulin (apolysaccharide found in plants) to the solid matrix, greatly increasesthe percentage of the applied DNA that can be eluted from the solidmatrix. This is particularly apparent when purified DNA is applied thesolid matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of inulin.

DETAILED DESCRIPTION OF THE INVENTION

A range of chemicals was added to a solid matrix to study their effecton the yields of DNA recovered from the solid matrix. In particular thesolid matrix known as FTA Elute (Whatman) has proved to be particularlyuseful in the practice of the current invention. However, it isanticipated that other types of solid matrix can also be used with theinvention. Many of the solid matrices are based on cellulose. The solidmatrix was treated with a solution of test reagents diluted in 2Mguanidine isothiocynate. Many chemicals had little or no effect on theyield of DNA recovered. Polyethylene glycol (PEG) at concentrations ofabout 10% had a small increase on the amount of DNA recovered. PEG is along chain polymer of ethylene glycol subunits. PEG is prepared in avariety of molecular weights defined by the average number of subunitsper molecule. Polymers of MW 400, 1000 and 3350 were evaluated. It wasobserved that PEG 1000 produced the best recovery of applied DNA results(data not shown) and was used in the remainder of the tests but isreferred to as PEG. At concentration of PEG at about 25% the resultsvaried between experiments; this may imply small inconsistencies in thecoating process of the solid matrix at these concentrations.

It was found that when the solid matrix was treated with the plantpolysaccharide inulin then increases in the amount of DNA eluted fromthe solid matrix was observed. It was found that adding inulin up toconcentrations of 20% to the solid matrix increases the yield of DNArecovered from the solid matrix from 25-40%, without the addition ofinulin to 80%. There were indications that adding 10% PEG in addition tothe added inulin increased the yield of recovered DNA to approximately85%.

Inulin is a naturally occurring polysaccharide found in many plants. Itsstructure is given in FIG. 1 . it is anticipated that simplemodifications of inulin eg esterification would be possible and stillachieve the improved elution of the applied purified DNA.

The purified DNA can be applied to the solid matrix that has beentreated with inulin in buffers that are routinely used in nucleic acidchemistry. Up to 10% PEG can also be included in the application buffer.The DNA prior to application to the solid matrix can be purified by avariety of standard laboratory techniques.

An important consideration is that the increased yield of recovering DNAis maintained with time ie. prolonged storage at room temperature. Ithas been found that DNA can be recovered with increased yield for atleast twenty-three days. It is expected that this increased yield willoccur with even longer storage periods. Room temperature is usuallyabout 20°-25° C. with a typical value of 20° C.

EXAMPLES

The present example is provided for illustrative purposes only, andshould not be construed as limiting the present invention as defined bythe appended claims.

Example 1 Matrix Chemistry Modification

The solid matrix was FTA Elute 903 matrix from Whatman.

-   -   1) A 4 M stock of guanidine thiocyanate was prepared and diluted        to 2 M using various concentrations of test reagents.    -   2) 903 matrix (2¼″×2¼″) was placed into trays containing        guanidine thiocyanate/test reagent mixes and agitated gently for        10 seconds.    -   3) Matrices were dried for 10 min on a metal rack using two hair        dryers (Simply Basic DS-727); one placed at a 30° angle 15 cm        above the matrix, the other placed 25 cm below the matrix at a        30° angle such that the two hair dryers and the matrix were in        alignment. Matrices were dried further without the air flow at        21±2° C. overnight.    -   4) Matrices were stored at room temperature in a desiccator        until use.

Example 2 Application of DNA to Test Solid Matrix

Human DNA (Roche) was spotted onto the test solid matrix atconcentration of 160 ng/μl. Usually a pre-punched 5 mm diameter disc ofthe matrix was used. Discs were dried at room temperature for a minimumof 3 hours.

Example 3 Elution of DNA from Solid Matrix

-   -   1) Each dried disc was placed in a sterile 1.5 ml microfuge tube        and washed with 500 μl dH₂O by pulse vortexing three times for a        total of five seconds.    -   2) Discs were transferred to 0.5 ml microfuge tubes containing        100 μl of dH₂O, ensuring that discs were fully submerged.    -   3) Microfuge tubes were placed in a heat block for 30 min at 98°        C.    -   4) Microfuge tubes were pulse vortexed for 60 sec and then        briefly centrifuged.    -   5) Eluates were transferred to new 0.5 ml tubes, leaving discs        behind. Eluates were stored at 4° C. until quantification.

Example 4 Quantification of DNA in Eluates

Quantification of DNA in eluates was performed by QPCR using a 7900HTThermal Cycler (Applied Biosystems). Reactions were set up using anRNase P assay and TAQMAN® Universal PCR Master Mix (Applied Biosystems).A four point standard curve was prepared using a serial dilution from 10to 0.01 ng/μl of the same Roche DNA used for experiments. Early QPCRquantifications were performed in 96-well plates and were set upmanually. Following the introduction and validation of a liquid handlingrobot, later quantifications were performed using 384-well plates. Both96 and 384-well plates were validated and also tested against each otherto check for consistency between methods.

Results

Results (Table 1) show that matrices impregnated with either inulin orPEG did result in increased DNA recovery compared to FTA Elute by itselfIn addition to this, the use of a spotting buffer containing 10% PEGresulted in an additional increase in DNA yield (85% when used with FTAElute+20% inulin).

TABLE 1 Percent recoveries of DNA (1 μg) applied to FTA Elute matricesimpregnated with additional chemicals. Spotting buffers were mixed withDNA immediately prior to application to pre-punched 5 mm diameter discs.For each entry, n = 4. Discs were dried and DNA eluted as described insection 1.1. Spotting Buffer DNA Only 10% PEG FTA Elute Matrix 10% PEG49 66 20% Inulin 62 85 FTA Elute 42 48

The matrix containing 20% inulin showed the highest % recovery ofapplied purified DNA. Since PEG was also identified as a possibleadditive to matrix impregnation chemistry, FTA Elute impregnated with acombination of 20% inulin and 10% PEG was also prepared for furtherinvestigation. A spotting buffer containing 10% PEG was confirmed asfurther increasing yields when used in conjunction with the modifiedmatrix chemistry.

Results were also obtained from experiments where the discs with appliedpurified DNA had been stored in a dessicator at room temperature. Theresults showed that the discs could be stored for at least twenty-threedays before DNA elution with similar increased recovery of DNA when thematrix had been treated with inulin.

Results also showed that the amount of DNA applied and recovered fromthe test matrix could be as low as 1 μg and as high as 1 μg (the maximumtested) and the increased effect on inulin treatment was still observed.

It is to be understood that any feature described in relation to any oneembodiment may be used alone, or in combination with other featuresdescribed, and may also be used in combination with one or more featuresof any other of the embodiments, or any combination of any other of theembodiments. Furthermore, equivalents and modifications not describedabove may also be employed without departing from the scope of theinvention, which is defined in the accompanying claims.

What is claimed is:
 1. A method of purifying a nucleic acid from acomposition, comprising: a) impregnating a solid matrix with a solutioncomprising inulin, polyethylene glycol (PEG), and a denaturant; b)applying the composition comprising the nucleic acid onto the solidmatrix impregnated with the solution comprising inulin, PEG, and thedenaturant; and c) eluting the nucleic acid from the solid matrix,thereby purifying the nucleic acid from the composition, whereinsuitable concentrations of inulin and PEG are obtained to yieldsynergistic amount of purified nucleic acid.
 2. The method of claim 1,wherein the solution has up to 10% by weight PEG.
 3. The method of claim1, wherein the composition further comprises a buffer comprising PEG. 4.The method of claim 2, wherein the PEG is in the solution at aconcentration of about 10% by weight.
 5. The method of claim 1, whereinthe denaturant is guanidine thiocyanate.
 6. The method of claim 1,wherein the matrix is a cellulose-based matrix.
 7. The method of claim1, wherein the solution comprises inulin in a concentration of up to 20%by weight, and PEG in a concentration of up to 10% by weight.
 8. Themethod of claim 1, further comprising drying the solid matrix at roomtemperature before the eluting.
 9. The method of claim 1, wherein themethod increases a yield of purified nucleic acid eluted from the solidmatrix by at least double compared to a yield of purified nucleic acideluted from a control solid matrix that has not been impregnated withthe solution.
 10. The method of claim 9, wherein the increased yield ofpurified nucleic acid eluted from the solid matrix is about 80% byweight.
 11. The method of claim 1, further comprising storing thenucleic acid on the solid matrix before the eluting.
 12. The method ofclaim 11, wherein the nucleic acid is not lyophilized.
 13. The method ofclaim 1, wherein the nucleic acid is DNA.
 14. The method of claim 1,wherein the nucleic acid is human DNA.